Several enzyme immunoassay test kits are commercially available for screening of matrices including cattle feed, tissues, faeces and urine for the presence of beta-agonists. The kits utilized for this evaluation offer sensitivity as low as 0.1 ng ml-1 (assay solution concentration, not test sample). Evaluation of ractopamine hydrochloride (LY31537) solutions at concentrations as high as 1000 ng ml-1, using six different kits from four different manufacturers, showed cross-reactivity of less than 0.5%.
A rapid, competitive particle concentration fluorescence immunoassay (PCFIA) for the determination of tylosin (Tylan) concentrations in extracts of pre-mix, feeds (cattle, chicken, and swine), and liquid feed supplement is described. Tylosin-β-phyco-erythrin conjugate, tylosin standard or diluted sample extracts, and rabbit anti-tylosin antibody are incubated for 15 min in PCFIA plates. Rabbit anti-tylosin antibody is then captured, during a 15 min incubation, with goat anti-rabbit antibody that is attached to latex beads. Assay plates are washed to remove unbound tylosin-(β-phyco-erythrin and unbound, free tylosin. Fluorescence is measured with a fluorimeter. Tylosin concentration is inversely proportional to the β-phycoerythrin fluorescence signal. The method, evaluated and validated with an IDEXX Screen Machine, shows high specificity with regard to compounds expected to be found in the presence of tylosin, and results correlate well with traditional turbidimetric assay results. Hydrolysis of TUA (tylosin-urea ad-duct), necessary with microbiological methods, and cleanup of extracts beyond filtration are unnecessary with the PCFIA for tylosin.
Sampling an animal feed containing a granulated analyte is extremely difficult. A granulated feed additive, Tylan® (Elanco, tylosin), was used to demonstrate how a granulated product can increase assay variability if steps are not taken to reduce and control this variability. The number of tylosin granules was determined for Granulated Tylosin Concentrate, the active ingredient of Tylan Premix. Multiple weighings of 3 lots of the concentrate were prepared, and the average number of granules per gram of concentrate was determined with the aid of image analyses. The total weight of Tylan Premix in varying analytical samples was calculated. Finally, the average number of tylosin granules per analytical sample was calculated based on the average number of granules per gram. A10 g analytical sample obtained from an animal ration containing 8 ppm tylosin activity would contain an average of 1.16 granules of tylosin, and a 100 g analytical sample would contain 11.6 granules. These calculations demonstrate the need to increase the analytical sample size in analyzing an animal feed containing a granulated feed additive.
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